Elucidating Membrane-Bound Transcription Regulation in Vibrio Cholerae via Single-Molecule Imaging

Abstract

dynamics of several replisome components in vivo. Photoactivated localization microscopy (PALM) and single-molecule tracking give a resolution of 20-40 nm, far below the diffraction limit of conventional microscopy, enabling us to localize and track every single protein molecule. In our study, we investigated the dynamics of a number of replisome components under different conditions, including the DNA polymerases PolC, and the b-clamp loader DnaX. We can watch the real time behavior of different replisome components during the DNA synthesis process, and study them quantitatively. Surprisingly, our investigations have revealed that all these replisome components are highly dynamic and exchange more rapidly than previously expected, and we characterize the molecular scale distribution of each replisome component within the dynamic replication machinery.

Nanoscopic Cellular Imaging: Confinement Broadens Understanding

“…Recently, CPD investigations of the virulence regulation protein TcpP labeled with the photoactivatable fluorescent protein PAmCherry at the native chromosomal locus in V. cholerae revealed a faster diffusing population that was not previously captured with ectopic expression (red curve in Figure 3d). 84 bound transcription regulation in V. cholerae can now be investigated. Overall, bacteria are among the most successful and widespread organisms on Earth, and their rapid growth rates, simple growth media, and established genetics make these organisms ideal to understand life's essential processes.…”